Many substances occur in body fluids and tissues, which are capable of binding to a specific binding partner but which themselves cannot trigger an immunological reaction and are therefore denoted haptens, which serve as parameters for certain diseases or for the state of health of the human body. Haptens include metabolites, hormones, neurotransmitters, lipoproteins, tumor markers and viral proteins among others. In addition, most drugs whose determination is often necessary for monitoring drug treatment are grouped with the haptens. Since all these haptens only occur in very small amounts one uses methods based on immunoassays for their detection. The various immunological methods of determination may be classified into homogeneous and heterogeneous methods. A solid phase reaction is always involved in the heterogeneous methods in order to separate the bound fraction of the labelled components from the unbound. In this type of method the label can be easily determined. A disadvantage is, however, that the heterogeneous reaction takes a long time and several steps of washing and separation.
In the homogeneous method variant there is no separation of bound label and unbound label and, as a result, differentiation between bound and unbound label has to take place by other methods. There are different possibilities for this. Thus, conjugated enzymes to e.g., can be used as label which only then become enzymatically active when they are bound to the hapten or antigen to be determined or when they are activated by the substance to be determined. A further possibility is to use a fluorescent substance as label whose fluorescence is either shifted to another wavelength range by binding to the substance to be determined or its polarization is changed.
A particular disadvantage of these known methods is that the sample often contains components which interfere with the test, thus necessitating pretreatment of sample in order to remove these substances. In addition, extensive optimization is necessary for each parameter, e.g. the enzymes must be modified in a way which depends on the parameter. In all these tests there are conflicting requirements for optimal differentiation and optimal sensitivity, since on the one hand the concentration of the particulate reagent should be limited in order to allow an adequate competitive reaction with the sample and on the other hand the particulate reagent should be highly concentrated and highly labeled in order to achieve an adequate signal change per unit time. The balance of these requirements leads to limited sensitivity and susceptibility to interference which can often only be eliminated by specific sample pre-treatment.
In order to solve these problems a homogeneous method of determination was suggested in European Patent-A0349 988 in which the sample solution is incubated with 3 receptors R1, R2 and R3 of which R1 and R2 are capable of binding to one another and R3 is capable of specific binding to the substance to be determined in which receptor R1 is a conjugate of a partner of a specific binding pair P and a substance S which corresponds to the substance to be determined or is a derivative thereof or at least has an epitope of the substance to be determined, R2 is a receptor which has at least two binding sites for the specific binding partner and R3 is a receptor which has at least two binding sites of which at least one binds specifically to an epitope of the substance to be determined or of S. On incubation of the sample solution with these three receptors the substance to be detected competes with the receptor R1 for binding to receptor R3 and receptor R2 binds with receptor R1. An agglutination results which can be monitored photometrically only when receptors R1, R2 and R3 bind. Binding of the substance to be determined to receptor R3 prevents the agglutination and therefore the agglutination is an indirect measure for the content of the substance to be detected. This method is suitable for the detection of immunologically active substances such as antigens, antibodies and haptens. For the detection of haptens, a conjugate of a partner of a specific binding pair and of a hapten is used as receptor R1. In a particularly advantageous embodiment a conjugate of biotin and substance S is used as receptor R1, latex coated with streptavidin (SA) is used as receptor R2 and an antibody capable of binding to the substance to be detected is used as receptor R3. The biotin (Bio)-hapten conjugate binds via the biotin moiety to the streptavidin-coated latex. The antibody can bind to the hapten-biotin conjugate via the hapten moiety. If two complexes of streptavidin-coated latex and biotin-hapten conjugate now bind to the antibody, turbidity then occurs which can be evaluated. The turbidity in this process occurs the more slowly the larger the amount to be analyzed and the smaller the solvation of the conjugate.
SA is a tetrameric protein isolated from Streptomyces avidinii with molecular weight about 60 kDa. Biotin, a 244 Da vitamin, binds with high affinity to SA. It is the strongest known non-covalent biological interaction (Ka≈10−15M−1). The SA-Bio complex is very rapidly formed and, once formed, is unaffected by external factors. Conjugates of a hapten and biotin have to be provided for the detection of haptens in the type of methods described above. Hapten-biotin conjugates have in fact been known for a long time. Thus in European Patent-A35 317 a so-called bidentate conjugate is described which consists of an immunologically active molecule and a specific binding partner which are linked together via a spacer. Investigations have been carried out in order to determine the extent to which the length of the spacer has an effect on the properties of the conjugate. As a result it was established in this literature reference that the conjugates have an optimal effectiveness when the spacer length is more than 22.2 Å, which corresponds approximately to a chain length of 18 atoms, but that, on the other hand, a chain length of more than 20 atoms reduces the sensitivity. In addition it is stated that the presence of more than 5 heteroatoms is disadvantageous. However, absolutely satisfactory results have not yet been achieved with these conjugates.
Additionally, it is known that bio-conjugation is a burgeoning field of research. Biological molecules are often coupled to other molecules or compounds for use in bioanalytical or biopharmaceutical applications. The covalent combination of a biological molecule and another molecule or compound is generally referred to as a “conjugate.” Novel methods for the mild and site-specific derivatization of small molecules, proteins, DNA, RNA, and carbohydrates have been developed for many applications such as ligand discovery, disease diagnosis, and high-throughput screening. These powerful methods owe their existence to the discovery of chemo-selective reactions that enable bio-conjugation under physiological conditions—a tremendous achievement of modern organic chemistry.
Bioanalytical or biopharmaceutical applications often require that compounds and biological molecules be coupled to other compounds or molecules to form a conjugate. For example, “immunoconjugate” generally refers to a conjugate composed of an antibody or antibody fragment and some other molecule such as a label compound (e.g., a fluorophore), a binding ligand (e.g., a biotin derivative), or a therapeutic agent (e.g., a therapeutic protein or toxin). These particular conjugates are useful in reporting the presence of the antibody, binding or capturing the antibody, and targeting the delivery of a therapeutic agent to a specific site, respectively. Depending upon a conjugate's use, a wide variety of conjugates may be prepared by coupling one conjugate component to another via a linker. Virtually an endless number of combinations of a biological molecule coupled to a label compound, binding ligand or therapeutic agent have been joined to create conjugates suitable for a particular purpose or need.
Typically, conjugates are prepared by covalently coupling one of the conjugate components to the other. For example, the immunoconjugate referenced above may be prepared by coupling a label compound, a binding ligand, or a therapeutic agent to an antibody or antibody fragment. Often the coupling involves the use of a linker compound or molecule which serves to join the conjugate components. Typically, the linker is selected to provide a stable coupling between the two components, and to control the length and/or the geometry over which the interaction can occur.
For example, biotin conjugates are widely used in biological sciences. Biotin is a naturally occurring vitamin which has an extremely high binding affinity for avidin and streptavidin. Because of the affinity of biotin for avidin, biotin-containing conjugates have been widely used in bioanalytical procedures including immunoassays, affinity chromatography, immunocytochemistry, and nucleic acid hybridization. Bioanalytical assays often take advantage of the high binding affinity of biotin for avidin through the covalent coupling of biotin to one of the assay components. Biotin may be covalently coupled to many different types of molecules, including proteins, such as antibodies, antibody fragments, enzymes and hormones; nucleic acids such as oligonucleotides and a nucleic acid probes; and smaller molecules such as drugs or other similar compounds. Moreover, in some applications biotin may be coupled to a solid phase or support. The covalent coupling of biotin to another molecule involves bond formation through chemical reaction between suitable chemical functional groups and a reactive biotin derivative. Reactive biotin derivatives for conjugation can be prepared from biotin, and are most commonly carboxylic acid derivatives, amines, or hydrazide derivatives. Common reactive biotin derivatives include reactive biotin esters such as an N-hydroxysuccinimide (NHS) ester, and biotin hydrazide. Alternatively, reactive biotin derivatives can be obtained from commercial sources including Sigma (St. Louis, Mo.), Pierce (Rockford, Ill.), Molecular Biosciences (Boulder, Colo.), and Molecular Probes (Eugene, Oreg.). Methods of conjugating biotin derivatives to proteins have been described in numerous publications (Harlow and Lane, Antibodies: A Laboratory Manual, NY: Cold Spring Harbor Laboratory, 1988, pp. 340-341, and Rose et al., Bioconjug. Chem. 2:154, 1991).
In addition to biotin, other compounds are commonly coupled to biological molecules for use in bioanalytical procedures. Typically, these compounds are useful in labeling the biological molecule for detection purposes. Common labeling compounds include fluorescent dyes, as well as ligands for binding to their respective binding partners. Examples of common fluorescent dyes used for this purpose include fluorescein and rhodamine, and examples of ligands for binding to their binding partners include drug compounds such as digoxigenin or digoxin and β-lactam antibiotics. Numerous other compounds suitable for use as labels in specific binding techniques have also been described in the literature. Like biotin, these compounds are generally derivatized to contain functional groups that react readily with the biological molecule. For example, fluorescein isothiocyanate is a reactive fluorescein derivative which may readily be conjugated to proteins through their sulfhydryl groups. Furthermore, the attachment of a tether containing thiol or polyhistidine functionalities allows a molecule of interest to be bound to a solid surface, such as, gold or nickel surfaces.
Effective conjugation of a compound, such as biotin or a fluorescent dye, to a biological molecule generally requires that the resulting labeled conjugate retain the bioactivity of the biological molecule. A conjugate may have only limited or no utility if, upon coupling, the functional activity of the biological molecule is diminished or lost. For example, for an antibody conjugate, retention of antigen binding activity (immunoreactivity) is of foremost importance. Because some antibodies lose immunoreactivity upon labeling of their free amino groups, presumably due to the presence of these groups in the antigen binding site of the antibody, the site or sites at which a label is attached to a biological molecule is of considerable importance. Similarly, some enzymes contain free amino groups in their active sites which, upon their use as a labeling site, may result in a loss of enzymatic activity. Many enzymes also contain sulfhydryl groups in their active sites and are inactivated by labeling with sulfhydryl-reactive compounds such as fluorescein isothiocyanate.
In addition to retaining bioactivity, the stability of the conjugate with respect to linkage of the compound to the biological molecule is also important. For example, loss of a label from a conjugate typically results in the loss of ability to follow the conjugate in a bioanalytical procedure. In an attempt to provide stable linkages, conjugates are often coupled through amide and hydrazone bonds. Amide linkages are formed by reaction between an amino group and a carboxylic acid group, and hydrazone linkages result from reaction of a carbonyl group (such as an aldehyde group) and a hydrazine group. The relatively high stability of these linkages at neutral pH has led to their wide use in conjugation techniques. However, these linkages are not flexible enough to allow control over the distance between the components and to control the hydrophobicity and hydrophilicity of the conjugates. In addition to amide linkages, other functional groups may be employed to couple the molecule of interest and the linkers. For example, alcohols and phenols can be coupled via ether or urethane groups, amines can be alkylated or converted to ureas, aryl halides can be linked by various carbon-carbon coupling methods, e.g. Heck or Stille coupling.
Accordingly, there is a need in the art for improved linkages for conjugating a biological small molecule with, for example, a label compound, a binding ligand or agent, or a therapeutic agent. Such linkages preferably have enhanced stability and control the length between the biological molecules.